A human papillomavirus (hereinafter referred to as “HPV”) is a virus having circular double-stranded DNA as genome, which induces proliferative lesions. HPV is classified into 100 or more kinds of subtypes. Also, it is known that the subtypes of HPV share common regions, which are nonstructural protein-coding E1 region, E2 region, E4 region, E5 region, E6 region and E7 region, capsid protein-coding L1 region and L2 region, and LCR.
HPV DNA has been detected in lesion sites of uterine cervical cancer and uterine cervical dysplasia, and tissues of oral cancer and pharyngeal cancer. Thus, HPV infection is regarded as one of the risk factors for uterine cervical cancer, oral cancer, and pharyngeal cancer. In most cases, a pattern of HPV infection is a transient infection in which HPV is spontaneously disappeared from a cell after a certain period of time from the establishment of infection. However, in 5 to 10% of HPV infections, there are cases where HPV is not disappeared and the infection turns into a persistent infection causing uterine cervical cancer.
It is to be noted that, in tissue diagnosis of uterine cervix, uterine cervical dysplasia is classified into three stages, namely mild-grade, moderate-grade, and high-grade dysplasia as a preliminary stage of generation of a cancer cell, depending on the degree of appearance of an atypical cell in the epithelium. When high-grade dysplasia is further aggravated, lesions of uterine cervical dysplasia reach a stage at which a cancer cell emerge in the epithelium. Then, uterine cervical dysplasia progresses to “intraepithelial carcinoma”, in which cancer cells are confined to the epithelium, and “microinvasive squamous cell carcinoma” and “invasive squamous cell carcinoma”, in which a cancer cell infiltrates from the epithelium down into subcutaneous tissues.
In most cases, lesions of mild-grade dysplasia or moderate-grade dysplasia are observed without particularly providing any treatment thereto. However, if a precursor lesion of high-grade dysplasia is left untreated, the lesion is highly likely to progress to invasive cancer. Thus, in many cases, treatment such as surgery is provided to a subject diagnosed as having high-grade dysplasia. Accordingly, determining whether or not a lesion in a subject is at a stage of high-grade dysplasia or a more severe stage is important in deciding a treatment method for the subject.
In chromosomal DNA in higher eukaryotes, among bases constituting DNA, the 5-position of cytosine is occasionally methylated. The methylation of DNA in higher eukaryotes functions as an inhibitory mechanism on the expression of genetic information. Recently, a report indicating that the presence or absence of methylation in HPV genomic DNA is strongly associated with onset of cancer has been made.
For example, Patent Document 1 describes that, when E6 region and LCR of HPV contained in a uterine cervical cell of a patient are not methylated, the presence of a uterine cervical cancer cell is more strongly indicated. However, there are cases where unmethylated E6 region and unmethylated LCR are detected in a cell other than a uterine cervical cancer cell. Thus, it is difficult to detect a cancer cell caused by HPV in uterine cervical cells of a patient with high accuracy only by confirming the methylation state of E6 region and LCR.
Also, Non-Patent Document 1 describes that, in uterine cervical cancer, L1 region is strongly methylated, whereas LCR and E6 region are not methylated in HPV-18. In Non-Patent Document 1, a nucleic acid that has been treated with a bisulfite salt, which corresponds to HPV-18 genomic DNA, is sequenced, and the methylation state thereof is confirmed. However, confirming methylation of DNA by sequencing is time-consuming and involves cumbersome operations. Thus, it is difficult to detect a cancer cell caused by HPV in a sample collected from a subject in a simple manner.
[Non-Patent Document 1] Tolga Turan et al., Virology 349 (2006) pp. 175-183
[Patent Document 1] National Publication of International Patent Application No. 2006-522607